Human papillomaviruses (HPVs) are a group of non-enveloped DNA viruses that have evolved to exploit a specialized biological niche in epithelial tissues such as the skin or the genital mucosa. Although most HPV types are associated with clinically inapparent or benign symptoms, such as common skin warts, about a dozen sexually-transmitted HPV types cause nearly all cases of cervical cancer, as well as a substantial fraction of anal, genital and throat cancers. HPV-induced cervical cancer kills nearly 300,000 women per year worldwide, mostly in developing countries where access to cervical cancer screening is limited. A recently-licensed vaccine targeting two cancer-causing HPV types has helped focus public attention on the substantial risk these viruses pose to public health. Work in the Tumor Virus Molecular Biology Section is focused on the HPV virion. The virions of non-enveloped viruses are dynamic structures that must undergo a range of conformational changes during different stages of the virus life cycle. Virions must be flexible enough to allow encapsidation of the viral genome, yet stable enough to withstand environmental insults encountered during transmission between hosts. Virions must also become pliable enough to release the viral genome upon infection of a host cell. Although each of these steps in the virus life cycle requires discrete structural motifs that are typically well-conserved among members of a virus family, immunological pressure tends to select for virions in which conserved functional motifs are occluded from immunological recognition. Using HPV vector technologies and a range of proteomics tools, we seek to characterize conserved functional motifs of the virion, as well as their cellular binding targets. Work in the lab is aimed at elucidating the mechanics of virion assembly and infectious entry, as well as the basic cell biology that underpins these phases of the viral life cycle. However, our work also has translational goals. High-titer HPV-based gene transfer vectors (also known as HPV pseudoviruses) are beginning to show promise as genetic vaccine and gene therapy vehicles. Improved understanding of the assembly, infectivity and in vivo tropism of HPV vectors should improve their practicality for in vivo gene transfer applications. Antibodies targeting conserved virion structures have the potential to confer sterilizing immunity against a wide variety of HPV types. Thus, another translational goal of our work is to facilitate the ongoing development of improved vaccines that might confer protection against a broader range of tumorigenic HPV types than the currently available vaccine. During FY10 we completed work that elucidates the pattern of sulfur-based crosslinks that stabilize the HPV virion. Our insights in this area allowed us to generate fully mature recombinant virions that are more stable and also more regular than standard preparations made using our previously described methods. In collaboration with Benes Trus (NIH/CIT) and Alasdair Steven (NIH/NIAMS), we have generated high resolution computerized reconstructions of the fully mature virion. The improved resolution allows the assignment of atomic-level positions of the L1 capsid protein of HPV type 16. The improved structure should facilitate the further mapping of functional motifs on the virion surface and improved understanding of the dynamics of the virion structure during assembly and infectious entry. The high resolution structure has facilitated our recent efforts to identify L1 residues that mediate the binding of the virion to the cell surface receptor heparan sulfate. We also continued work aimed at identifying cellular proteins that facilitate the infectious entry of HPVs. We provided support for two collaborative publications that highlight our discovery that a cellular protease complex called gamma secretase plays an important role in HPV infectious entry. This work could lead to the development of protease inhibitor drugs that could block the infectious entry of HPVs when applied topically.